DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. In this work, I reported the completion of a transposon mutagenesis screen in budding yeast and the identification of five novel genes (RRD1, CIK1, CTF18, RTS1, and IRC6) critical for telomere healing. The characterization of Rrd1 led to the surprising finding that Rrd1 facilitates telomere healing at DSBs with little or no TG-rich sequences but not at DSBs with long tracts of telomeric sequences. Pph3, a PP4 phosphatase, acts in conjunction with Rrd1 to promote telomere healing. Conversely, Mec1, the ATR ortholog, phosphorylates Cdc13 on its S306 residue to suppress its accumulation at DSBs. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DSBs. Next, I found that Cik1 and its kinesin partner Kar3 are both important for telomere healing. Importantly, Kar3 contributes to telomere healing through its motor function. In contrast to Rrd1, Kar3 contributes to telomere healing regardless of telomeric sequence lengths adjacent to the break. Finally, Cik1 and Kar3 have a general role in DNA repair and physically associate with DSBs, which is dependent on the process of anchoring DSBs to nuclear periphery. In conclusion, I identified a mechanism by which the ATR family of kinases enforces genome integrity, a phosphoregulatory loop that underscores the contribution of Cdc13 to the fate of DNA ends, and a kinesin complex critical for the spatial organization of DNA repair.

Kernel patches are frequently released to fix security vulnerabilities and bugs. However, users and system administrators often delay installing these updates because they require a system reboot, which results in disruption of service and the loss of application state. Unfortunately, the longer an out-of-date system remains operational, the higher is the likelihood of a system being exploited. Approaches, such as dynamic patching and hot swapping, have been proposed for updating the kernel. All of them either limit the types of updates that are supported, or require significant programming effort to manage. We have designed a system that checkpoints application-visible state, updates the kernel, and restores the application state. By checkpointing high-level state, our system no longer depends on the precise implementation of a patch and can apply all backward compatible patches. The results show that updates to major kernel releases can be applied with minimal changes.

DNA damage represents a major challenge to the faithful replication and transmission of genetic information from one generation to the next. Cells utilize a highly integrated network of pathways to detect and accurately repair DNA damage. Mutations arise when DNA damage persists undetected, unrepaired, or repaired improperly. Mutations are a driving force of carcinogenesis and therefore many of the DNA damage surveillance and repair mechanisms guard against the transformation of normal cells into cancer cells. Central to the detection and repair of DNA damage is the relocalization of DNA damage surveillance proteins to DNA damage where they assemble into subnuclear foci and are capable to producing a signal that the cell interprets to induce cellular modifications such as cycle arrest and DNA repair which are important DNA damage tolerance. In this work, I describe my quest to understand the mechanisms underlying the assembly, maintenance, and disassembly of these DNA damage-induced foci and how they affect DNA damage signaling in Saccharomyces cerevisiae. First, I describe phenotypic characterization of a novel mutation that impairs assembly of the 9-1-1 checkpoint clamp complex into foci. Second, I describe my work to further understand the roles of the histone phosphatase Pph3 and phosphorylated histone H2A in modulating DNA damage signaling. Third, I include my work to uncover the possible mechanism by which the helicase Srs2 works to enable termination of DNA damage signaling. In summary, this thesis documents my efforts to understand the cellular and molecular nature of DNA damage signaling and how signaling is turned off in coordination with DNA damage repair.

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. In this work, I reported the completion of a transposon mutagenesis screen in budding yeast and the identification of five novel genes (RRD1, CIK1, CTF18, RTS1, and IRC6) critical for telomere healing. The characterization of Rrd1 led to the surprising finding that Rrd1 facilitates telomere healing at DSBs with little or no TG-rich sequences but not at DSBs with long tracts of telomeric sequences. Pph3, a PP4 phosphatase, acts in conjunction with Rrd1 to promote telomere healing. Conversely, Mec1, the ATR ortholog, phosphorylates Cdc13 on its S306 residue to suppress its accumulation at DSBs. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DSBs. Next, I found that Cik1 and its kinesin partner Kar3 are both important for telomere healing. Importantly, Kar3 contributes to telomere healing through its motor function. In contrast to Rrd1, Kar3 contributes to telomere healing regardless of telomeric sequence lengths adjacent to the break. Finally, Cik1 and Kar3 have a general role in DNA repair and physically associate with DSBs, which is dependent on the process of anchoring DSBs to nuclear periphery. In conclusion, I identified a mechanism by which the ATR family of kinases enforces genome integrity, a phosphoregulatory loop that underscores the contribution of Cdc13 to the fate of DNA ends, and a kinesin complex critical for the spatial organization of DNA repair.

El checkpoint de la fase S constitueix una barrera anti-càncer. Quan aquest mecanisme de vigilància detecta situacions que posen en perill la integritat genòmica, tals com dany al DNA o estrès replicatiu, genera una resposta que engloba la protecció de la replicació, l'aturada del cicle cel·lular i l'activació i coordinació dels sistemes de reparació del DNA. En última instància, si la crisi no es superada el checkpoint promou l'entrada en apoptosis via p53. Per aquest motiu, la mutació d'elements d'aquest mecanisme de control ha estat àmpliament implicada en tumors primaris humans, ja que afavoreixen l'aparició d'inestabilitat genòmica, un dels motors de la transformació cel·lular maligna. A pesar de la importància d'aquest mecanisme de vigilància en el seu paper antitumoral, la gran majoria de les seves dianes romanen desconegudes. Per tal d'identificar noves seqüències implicades en la resposta, es van realitzar 2 aproximacions a gran escala, obtenció de proteomes 2D-DIGE checkpoint dependents utilitzant com a model S. cerevisiae i l'obtenció de microxips d'expressió en resposta a estrès replicatiu en cèl·lules HeLa. Finalment, van permetre identificar la ciclina E2 com a nova diana del checkpoint de fase S. La ciclina E2 és imprescindible perquè les cèl·lules pugin superar la transició G1/S, comprometent-se a una ronda de cicle cel·lular. En resposta a estrés genotòxic és acumulada de forma dependent de l'activació del checkpoint de la fase S. Aquesta estabilització implica alts nivells del seu mRNA i requereix síntesi de novo continuada. Els nostres resultats indiquen que l'acumulació de ciclina E2 podria actuar alentint la progressió de la fase S, evitant d'aquesta manera que cromosomes danyats siguin segregats en mitosis aberrants. Per altra banda, la ciclina E2 es troba sobreexpressada en un elevat número de tumors primaris. Donada la seva implicació en la resposta del checkpoint, vam avaluar si presenta modulació a la resposta a agents quimioteràpics que afecten l'estructura del DNA. Els assaigs de viabilitat in vitro enfront a inhibidors de la topoisomerasa I, mostren que la ciclina E2 confereix resistència enfront la teràpia. Per tant, de confirmar-se aquests resultats in situ, podria ser utilitzada com a biomarcador de resposta. / The S-phase checkpoint acts as an anti-cancer barrier. When the surveillance mechanism detects dangerous situations for the genome integrity such as DNA damage or replication stress, the S-phase checkpoint generates its own response, including replication protection, cell cycle arrest and activation and coordination of DNA repair systems. Finally, if stress situation is not by-passed, the S-phase checkpoint induces cell death through apoptosis via p53. For these reasons, mutations in the mechanism elements are widely related with tumorogenesis processes, because they arise the emergence of genome instability, a driving force of malignant cell transformation. Despite the importance of this mechanism in its anti-cancer role, most of its targets are still unknown. To identify new sequences involved in the response, we used two high-throughput approaches: checkpoint-dependent 2D-DIGE proteomes in S. cerevisiae under replication stress conditions and expression microarrays in replication stress conditions using human HeLa cells. Finally we identified cyclin E2 as a novel S-phase checkpoint target. Cyclin E2 is essential to cells to by-pass G1/S boundary, entering in cell cycle. Cyclin E2 levels are stabilized in a S-phase checkpoint dependent manner. This stabilization involves the cyclin E2 mRNA increase and the continuous de novo synthesis of the protein. Our results indicate that cyclin E2 accumulation could delay the S-phase progression, avoiding the segregation of partially replicated chromosomes in aberrant mitosis. On the other hand, as cyclin E2 has been found over-expressed in a high number of human primary tumours, we next tested if the over-expression of cyclin E2 could modulate the response to DNA-based chemotherapy agents. Viability in vitro assays confirmed that cyclin E2 confers resistance to toisomerase I inhibitors. This result postulates cyclin E2 tumour levels as a prediction biomarker for chemotherapy response.

Initiation of DNA replication requires the action of the Dbf4/Cdc7 kinase complex (DDK) which is also a phosphorylation target of Rad53 kinase in the S-phase checkpoint. DDK is thought to trigger DNA replication by phosphorylating members of the Mcm2-7 complex present at origins of replication. While DDK phosphorylation sites have been identified on Mcm2-7, the contributions made by Dbf4 and Cdc7 to the targeting of the complex have not been established. DDK has also been implicated in the S-phase checkpoint response since it is removed from chromatin in a Rad53-dependent manner. The interaction of Dbf4 and Cdc7 with each of the Mcm2-7 subunits was assessed and showed an interaction between Dbf4 and Mcm2 and Mcm6, while interactions between Cdc7 and Mcm4 and Mcm5 were observed. Mutations in Mcm2 and Mcm4 that disrupt the interactions with Dbf4 or Cdc7 showed modest growth impairment and compromised DNA replication, while simultaneous abrogation of both interactions resulted in lethality. Strains overexpressing Mcm2 or Mcm4 were sensitive to genotoxic agents, while overexpression of Mcm2 in a Mcm4Δ175-333 strain background resulted in a severe growth impairment as well as sensitivity to genotoxic stress. ChIP analysis revealed the possibility of Dbf4/Cdc7 localization to origin flanking regions through most of S-phase, which may redistribute to origins at the time of firing. Fluorescence microscopy of Mcm2 and Dbf4 in S-phase seem to show a punctate pattern of staining, consistent with these factors’ localization to ‘replication factories.’ By using a Dbf4ΔN mutant, the N-motif was shown to be required for the Rad53-mediated removal of Dbf4 from chromatin under checkpoint conditions. Initial optimization of a DNA combing protocol was also performed, which along with Dbf4ΔN mutant and the fluorescently-epitope tagged strains, will be useful tools for evaluating a role for DDK in the S-phase checkpoint response. Altered levels of DNA replication factors have been implicated in many human cancers. The data presented in this study provide novel insight into the normal process of the initiation of DNA replication which can be applied to research involving higher eukaryotes, including humans, and can serve as a benchmark for comparison with the cancer phenotype.

Hepatocarcinoma cancer (HCC) is one the most common visceral malignancies in Taiwan, which has a very high incidence and a devastatingly poor prognosis. BMP4, belonging to the TGF-£] super-family of proteins is a multifunctional cytokine, known to exert its biological effects through SMAD and non-SMAD dependent pathways and is also known to be involved in human carcinogenesis. However, the effects of the BMP4 signaling in liver carcinogenesis are not yet clearly defined. In this study, we first demonstrate that BMP4 and its receptor, BMPR1A, are over-expressed in a majority of primary HCC and promote the growth and migration of HCC cell lines in vitro. We also further identify that BMP4 can induce HCC CDK1 and cyclinB1 up-regulation to accelerate cell cycle progression. Our study indicates that the induction of HCC cell proliferation is independent on the SMAD signaling pathway, since Smad4 knockdown of BMP4 induced HCC cell lines still leads to the up-regulation of CDK1 and cyclinB1 expression in HCC. Using MEK kinase selective inhibitors, the induction of CDK1 and cyclinB1 mRNA and protein were shown to be dependent on the activation of MEK/ERK signaling. In vivo xenograft studies confirmed that the BMPR1A- knockdown cells were significantly less tumorigenic than control groups. Taken together, our findings show that the up-regulation of BMP4 and BMPR1A in HCC promote the proliferation and metastasis of HCC cells and that CDK1 and cyclinB1 are important, SMAD-independent molecular targets in BMP4 signaling pathways during the HCC tumorigenesis. We propose here that BMP4 signaling pathways may have potential as new therapeutic targets, in HCC treatment.

From microscopic bacteria to the giant whale, every single living organism on Earth uses the same language of life: DNA. Deoxyribonucleoside triphosphates––dNTPs (dATP, dTTP, dGTP, and dCTP)––are the building blocks of DNA and are therefore the “alphabet of life”. A balanced supply of dNTPs is essential for integral DNA transactions such as faithful genome duplication and repair. The enzyme ribonucleotide reductase (RNR) not only synthesizes all four dNTPs but also primarily maintains the crucial individual concentration of each dNTP in a cell. In this thesis we investigated what happens if the crucial balanced supply of dNTPs is disrupted, addressing whether a cell has a mechanism to detect imbalanced dNTP pools and whether all pool imbalances are equally mutagenic. To address these questions, we introduced single amino acid substitutions into loop 2 of the allosteric specificity site of Saccharomyces cerevisiae RNR and obtained a collection of yeast strains with different but defined dNTP pool imbalances. These results directly confirmed that the loop 2 is the structural link between the substrate specificity and effector binding sites of RNR. We were surprised to observe that mutagenesis was enhanced even in a strain with mildly imbalanced dNTP pools, despite the availability of the two major replication error correction mechanisms: proofreading and mismatch repair. However, the mutagenic potential of different dNTP pool imbalances did not directly correlate with their severity, and the locations of the mutations in a strain with elevated dTTP and dCTP were completely different from those in a strain with elevated dATP and dGTP. We then investigated, whether dNTP pool imbalances interfere with cell cycle progression and if they are detected by the S-phase checkpoint, a genome surveillance mechanism activated in response to DNA damage or replication blocks. The S-phase checkpoint was activated by the depletion of one or more dNTPs. In contrast, when none of the dNTP pools was limiting for DNA replication, even extreme and mutagenic dNTP pool imbalances did not activate the S-phase checkpoint and did not interfere with the cell cycle progression. We also observed an interesting mutational strand bias in one of the mutant rnr1 strains suggesting that the S-phase checkpoint may selectively prevent formation of replication errors during leading strand replication. We further used these strains to study the mechanisms by which dNTP pool imbalances induce genome instability. In addition, we discovered that a high dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions, which are difficult to bypass at normal dNTP concentrations. Our results broaden the role of dNTPs beyond ‘dNTPs as the building blocks’ and suggest that dNTPs are not only the building blocks of DNA but also that their concentrations in a cell have regulatory implications for maintaining genomic integrity. This is important as all cancers arise as a result of some kind of genomic abnormality.

Checkpoint kinase 1 (Chk1) is a key player in the DNA damage response signalling pathway and the mode of Chk1 activation whereby it undergoes ATRdependent phosphorylation at Ser317 and Ser345 is well characterised. It has been suggested that phosphorylation at the ATR sites relieves the auto-inhibitory action conferred by the C-terminal negative regulatory domain on the catalytic core of Chk1. In this study, we show that Chk1 activity can also be stimulated by docking to an N-terminal region of the growth regulator p21waf1 and this docking domain is necessary for efficient Chk1-dependent phosphorylation of p21 at Ser146. In addition, Chk1 and p21 are shown to form a transient interaction by immunoprecipitation. Interestingly, although the isolated p21 docking domain can activate Chk1 in trans, a mutant where the C-terminal 70 amino acids are truncated is refractory to stimulation whereas mutation of the ATR phosphoacceptor sites does not affect docking dependent activation. Furthermore, when the amino acid sequence of the p21 docking domain was aligned with the sequence of Chk1, homology to the F region on the kinase domain was identified. Mutation of two conserved tryptophan residues within the homology region appears to release the C-terminus from intramolecular interactions rendering it susceptible to cleavage and refractory to allosteric stimulation. Furthermore, small peptides based on this region of Chk1, like the p21 docking domain, are able to activate Chk1 in trans and disrupt interaction between the N-terminal and Cterminal domains. Interestingly, peptide microarray showed that Chk1 stimulated by activating peptide is able to phosphorylate novel peptide substrates which are not observed with unstimulated Chk1. The data suggest that the last C-terminal 70 amino acids of Chk1 play an important role in auto-inhibition through interaction with the F region of the core catalytic domain. Binding to p21 is able to activate Chk1 by inhibiting the auto-inhibitory interaction independent of phosphorylation at the Ser317 and Ser345 sites. Furthermore, activating peptide is able to modulate Chk1 specificity towards other substrates.

The cell cycle is a highly complex process that is under the control of several pathways.  Failure to regulate and/or complete the cell cycle often leads to cell cycle arrest, which may be followed by programmed cell death (apoptosis). One cell type that has a variety of unique cell cycle properties is the horizontal cell of the chicken retina. In this thesis we aimed to characterize the final cell cycle of retinal horizontal cells. In addition, the regulation of the cell cycle and the resistance to apoptosis of retinal horizontal cells are investigated. Our results show that the final cell cycle of Lim1-expressing horizontal progenitor cells is heterogenic and three different cell cycle behaviors can be distinguished. The horizontal cells are generated by: (i) an interkinetic nuclear migration with an apical mitosis; (ii) a final cell cycle with an S-phase that is not followed by mitosis, such cells remain with a fully or partially replicated genome; or (iii) non-apical (basal) mitoses. Furthermore, we show that the DNA damage response pathway is not triggered during the heterogenic final cell cycle of horizontal progenitor cells. However, chemically induced DNA damage activated the DNA damage response pathway without leading to cell cycle arrest, and the horizontal progenitor cells entered mitosis in the presence of DNA damage. This was not followed by apoptosis, despite the horizontal cells being able to functionally activate p53, p21CIP1/waf1, and caspase-3. Finally, we show that FoxN4 is expressed in horizontal progenitor cells and is required for their generation. Over-expression of FoxN4 causes cell death in several neuronal retinal cell types, except horizontal cells, where it results in an overproduction. In conclusion, in this thesis, a novel cell cycle behavior, which includes endoreplication not caused by DNA damage and a basal mitosis that can proceed in the presence of DNA damage, is described. The cell cycle and cell survival processes are of particular interest since retinal horizontal cells are suggested to be the cell-of-origin for retinoblastoma.